Investigation of the pathogens contributing to naturally occurring outbreaks of infectious bovine keratoconjunctivitis (pinkeye) using Next Generation Sequencing

Anis, Eman; Kattoor, Jobin Jose; Greening, Sabrina S.; Jones, Lee W.; Wilkes, Rebeca P.

doi:10.1016/j.vetmic.2023.109752

Veterinary Microbiology

2023

DOI: 10.1016/j.vetmic.2023.109752

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Investigation of the pathogens contributing to naturally occurring outbreaks of infectious bovine keratoconjunctivitis (pinkeye) using Next Generation Sequencing

Eman Anis

Jobin Jose Kattoor

Sabrina S. Greening

et al.

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Cited by 5 publications

(19 citation statements)

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“…Based on the side-by-side histograms, the IBK group tended to have a lower number of reads for M. bovoculi than for the non-IBK group, and the 2 distributions mostly overlap for M. oculi . Based on our previous findings, 5 M. oculi is likely a nonpathogenic Moraxella sp. that is part of the normal ocular microbiota of cattle.…”

mentioning

confidence: 77%

“…We identified another Moraxella species (suggested name M. oculi ) associated with both IBK and clinically healthy eyes. 5 In addition, more than one Moraxella spp. can be detected in a single pinkeye case.…”

mentioning

confidence: 99%

“…10 As well, the regions that are used routinely for speciation, such as 16S rRNA gene variable regions, 13 may fail to provide accurate results. 5,7 A multiplex PCR assay designed to detect and speciate both M. bovis and M. bovoculi using the cytotoxin A gene has been described. 16 However, that PCR assay has not been evaluated with M. ovis , an organism that is detected occasionally from bovine eyes.…”

mentioning

confidence: 99%

“…Then, a preliminary evaluation was performed with ocular swabs obtained for a separate study from 13 cattle that were age- and breed-matched (7 with pinkeye, 6 with clinically healthy eyes) from 2 separate farms known to have M. oculi based on culture and ITS Sanger sequencing. 5 The nucleic acid was extracted from the ocular swab collected from each animal (DNeasy blood and tissue kit; Qiagen) and stored at −80°C until tested. These samples had been found to contain Moraxella spp.…”

mentioning

confidence: 99%

“…based on a targeted 16S rRNA gene metagenomics approach using primers to amplify and sequence the V2, V4, V8, V3–6, and V7–9 hypervariable regions (Ion 16S metagenomics kit; Thermo Fisher). 5 Despite obtaining a genus-level identification for these samples and the potential for speciation based on sequencing multiple hypervariable regions, the 16S rRNA gene is a poor target for speciating Moraxella . 5 A previous study showed interspecies recombination within the 16S rRNA gene of Moraxella , which necessitates evaluation of other genetic segments of these organisms to accurately resolve to the species level.…”

mentioning

confidence: 99%

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Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs

Wilkes,

Kattoor,

Weng

et al. 2023

J VET Diagn Invest

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Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of Moraxella: M. bovis and M. bovoculi. A third novel Moraxella spp., designated tentatively as M. oculi, has been identified from the eyes of cattle with and without pinkeye. These 3 Moraxella spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessing Moraxella diversity found in IBK- and non-IBK–affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3 Moraxella spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3 Moraxella spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3 Moraxella spp. Targeted NGS with sequencing of Moraxella spp. housekeeping genes appears to be a suitable method for speciation of Moraxella directly from ocular swabs.

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confidence: 77%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs

Wilkes,

Kattoor,

Weng

et al. 2023

J VET Diagn Invest

Self Cite

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Moraxella oculi sp. nov., isolated from a cow with infectious bovine keratoconjunctivitis

Wilkes,

Anis,

Kattoor

2024

International Journal of Systematic and Evolutionary Microbiology

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A novel species of the genus Moraxella was isolated from an ocular swab from a cow with infectious bovine keratoconjunctivitis. 16S rRNA gene sequencing suggested this species was Moraxella bovis (99.59 % nucleotide identity). Average nucleotide identity was calculated using a draft whole genome sequence of this strain compared with type strains of closely related Moraxella species and results established that it represents a new species. The genome size was 2 006 474 nucleotides and the G+C content was 42.51 mol%. The species could not be identified by matrix assisted laser desorption/ionization–time of flight mass spectrometry using a commercial database, confirming the novelty of the strain. We propose the name Moraxella oculi sp. nov. for this new species. The type strain is Tifton1T and has been deposited into the American Type Culture Collection (TSD-373T) and the National Collection of Type Cultures (NCTC), UK Health Security Agency (NCTC 14942T).

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Alterations to the bovine bacterial ocular surface microbiome in the context of infectious bovine keratoconjunctivitis

Gafen,

Liu,

Ineck

et al. 2023

anim microbiome

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Background Infectious bovine keratoconjunctivitis (IBK) is a common cause of morbidity in cattle, resulting in significant economic losses. This study aimed to characterize the bovine bacterial ocular surface microbiome (OSM) through conjunctival swab samples from Normal eyes and eyes with naturally acquired, active IBK across populations of cattle using a three-part approach, including bacterial culture, relative abundance (RA, 16 S rRNA gene sequencing), and semi-quantitative random forest modeling (real-time polymerase chain reaction (RT-PCR)). Results Conjunctival swab samples were obtained from eyes individually classified as Normal (n = 376) or IBK (n = 228) based on clinical signs. Cattle unaffected by IBK and the unaffected eye in cattle with contralateral IBK were used to obtain Normal eye samples. Moraxella bovis was cultured from similar proportions of IBK (7/228, 3.07%) and Normal eyes (1/159, 0.63%) (p = 0.1481). Moraxella bovoculi was cultured more frequently (p < 0.0001) in IBK (59/228, 25.88%) than Normal (7/159, 4.40%) eyes. RA (via 16 S rRNA gene sequencing) of Actinobacteriota was significantly higher in Normal eyes (p = 0.0045). Corynebacterium variabile and Corynebacterium stationis (Actinobacteriota) were detected at significantly higher RA (p = 0.0008, p = 0.0025 respectively) in Normal eyes. Rothia nasimurium (Actinobacteriota) was detected at significantly higher RA in IBK eyes (p < 0.0001). Alpha-diversity index was not significantly different between IBK and Normal eyes (p > 0.05). Alpha-diversity indices for geographic location (p < 0.001), age (p < 0.0001), sex (p < 0.05) and breed (p < 0.01) and beta-diversity indices for geographic location (p < 0.001), disease status (p < 0.01), age (p < 0.001), sex (p < 0.001) and breed (p < 0.001) were significantly different between groups. Modeling of RT-PCR values reliably categorized the microbiome of IBK and Normal eyes; primers for Moraxella bovoculi, Moraxella bovis, and Staphylococcus spp. were consistently the most significant canonical variables in these models. Conclusions The results provide further evidence that multiple elements of the bovine bacterial OSM are altered in the context of IBK, indicating the involvement of a variety of bacteria in addition to Moraxella bovis, including Moraxella bovoculi and R. nasimurium, among others. Actinobacteriota RA is altered in IBK, providing possible opportunities for novel therapeutic interventions. While RT-PCR modeling provided limited further support for the involvement of Moraxella bovis in IBK, this was not overtly reflected in culture or RA results. Results also highlight the influence of geographic location and breed type (dairy or beef) on the bovine bacterial OSM. RT-PCR modeling reliably categorized samples as IBK or Normal.

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Investigation of the pathogens contributing to naturally occurring outbreaks of infectious bovine keratoconjunctivitis (pinkeye) using Next Generation Sequencing

Cited by 5 publications

References 23 publications

Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs

Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs

Moraxella oculi sp. nov., isolated from a cow with infectious bovine keratoconjunctivitis

Alterations to the bovine bacterial ocular surface microbiome in the context of infectious bovine keratoconjunctivitis

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